1. Field of the Invention
The present invention relates to structural variants of anti-CD20 antibodies and/or antigen binding fragments thereof, preferably involving the amino acid sequences of complementarity-determining regions (CDRs), with improved therapeutic characteristics. In particular embodiments, the structural variation may comprise changes to the third CDR sequence of the antibody heavy chain (CDRH3), for example substitution of an aspartate residue for an asparagine residue at Kabat position 101. In other particular embodiments, the structural variation may comprise an arginine residue at Kabat position 94, which may form a salt bridge with an aspartate at Kabat position 101. In still other particular embodiments, the structural variation may comprise a valine residue at Kabat position 102. Such structural variants may provide improved efficacy for diseases related to proliferation of B-cells, such as B-cell leukemias, lymphomas or autoimmune diseases, as well as other immune diseases implicating B-cells. In preferred embodiments, the improved efficacy may allow administration of low dosages of anti-CD20 antibody or antigen binding fragment thereof, such as 80 mg or less, more preferably 50 mg or less, most preferably 30 mg or less, which may be administered two or more times about one to three weeks apart, or even two or more times weekly.
The anti-CD20 antibody may be a humanized, chimeric or human anti-CD20 antibody, particularly a monoclonal antibody (MAb). Other embodiments may concern therapeutic and/or diagnostic conjugates of humanized, chimeric or human anti-CD20 antibodies and methods of treating B-cell lymphomas and leukemias and various autoimmune diseases, for example using humanized, chimeric or human anti-CD20 antibodies. Still other embodiments may relate to antibody fusion proteins or antigen binding fragments thereof comprising at least one anti-CD20 MAb or antigen binding fragment thereof, in some cases in combination with a second, different antibody, especially anti-CD20 antibody, preferably anti-CD20 MAb, or antigen binding fragment thereof. The humanized, chimeric or human MAbs, antigen binding fragments thereof or antibody fusion proteins may be administered alone, as a therapeutic immunoconjugate or in combination with one or more therapeutic agents, with other naked antibodies or other immunoconjugates. Still other embodiments relate to DNA sequences encoding humanized, chimeric or human anti-CD20 antibodies and antibody fusion proteins, vectors and host cells containing the DNA sequences, and methods of making the humanized, chimeric or human anti-CD20 antibodies.
2. Background
The immune system of vertebrates consists of a number of organs and cell types which have evolved to accurately recognize foreign antigens, specifically bind to, and eliminate/destroy such foreign antigens. Lymphocytes, amongst others, are critical to the immune system. Lymphocytes are divided into two major sub-populations, T cells and B cells. Although inter-dependent, T cells are largely responsible for cell-mediated immunity and B-cells are largely responsible for antibody production (humoral immunity).
In humans, each B-cell can produce an enormous number of antibody molecules. Such antibody production typically ceases (or substantially decreases) when a foreign antigen has been neutralized. Occasionally, however, proliferation of a particular B-cell will continue unabated and may result in cancers known as B-cell lymphomas or leukemias. B-cell lymphomas, such as the B-cell subtype of non-Hodgkin's lymphoma, are significant contributors to cancer mortality. The response of B-cell malignancies to various forms of treatment is mixed. For example, in cases in which adequate clinical staging of non-Hodgkin's lymphoma is possible, field radiation therapy can provide satisfactory treatment. Still, about one-half of the patients die from the disease. Devesa et al., J. Nat. Cancer Inst. 79:701 (1987).
The majority of chronic lymphocytic leukemias are of B-cell lineage. Freedman, Hematol. Oncol. Clin. North Am. 4:405 (1990). This type of B-cell malignancy is the most common leukemia in the Western world. Goodman et al., Leukemia and Lymphoma 22:1 (1996). The natural history of chronic lymphocytic leukemia falls into several phases. In the early phase, chronic lymphocytic leukemia is an indolent disease, characterized by the accumulation of small mature functionally-incompetent malignant B-cells having a lengthened life span. Eventually, the doubling time of the malignant B-cells decreases and patients become increasingly symptomatic. While treatment can provide symptomatic relief, the overall survival of the patients is only minimally affected. The late stages of chronic lymphocytic leukemia are characterized by significant anemia and/or thrombocytopenia. At this point, the median survival is less than two years. Foon et al., Annals Int. Medicine 113:525 (1990). Due to the very low rate of cellular proliferation, chronic lymphocytic leukemia is resistant to cytotoxic drug treatment. Both chronic and acute lymphocytic leukemias of B-cell origin are suitable targets for the therapies described herein.
Traditional methods of treating B-cell malignancies, including chemotherapy and radiotherapy, have limited utility due to toxic side effects. The use of monoclonal antibodies to direct radionuclides, toxins, or other therapeutic agents offers the possibility that such agents can be delivered selectively to tumor sites, thus limiting toxicity to normal tissues. Also, the presence of B-cell antigens on these B-cell malignancies makes them optimal targets for therapy with unconjugated B-cell antibodies, such as against CD19, CD20, CD21, CD23, and CD22 markers on B-cells. HLA-DR, CD30, CD37, CD40, CD45, CD70, CD79a, and other antigens may serve as targets for normal and malignant B-cells, although they are also expressed on other cell types. Further, certain MUC1, MUC2, MUC3, and MUC4 antigens, preferably MUC1, as well as also insulin-like growth factors (ILGF), insulin-like growth factor receptor, macrophage migration-inhibitory factor (MIF), are also expressed in different hematopoietic malignancies, including B-cell tumors expressing CD20 and other B-cell markers. Still other antigen targets, such as those associated with the vascular endothelium of tumors, including tenascin, vascular endothelium growth factor receptor (VEGFR), and placental growth factor (P1GF), as well as other categories of antigens associated with B-cell malignancies, such as oncogene products (cMET, Kras, bcl-2, bcl-6), are also suitable targets for therapeutic antibodies.
B-cells comprise cell surface proteins which can be utilized as markers for differentiation and identification. One such human B-cell marker is the human B lymphocyte-restricted differentiation antigen, Bp35, referred to as CD20. CD20 is expressed during early pre-B-cell development and remains until plasma cell differentiation. CD20 is expressed on both normal B cells and malignant B cells whose abnormal growth can lead to B-cell lymphomas and leukemias. Antibodies against the CD20 antigen have been investigated for the therapy of B-cell lymphomas and leukemias. For example, a chimeric anti-CD20 antibody, designated as “IDEC-c2B8” (rituximab), has activity against B-cell lymphomas when provided as unconjugated antibodies at repeated injections of doses exceeding 500 mg per injection. Maloney et al., Blood 84:2457 (1994); Longo, Curr. Opin. Oncol. 8:353 (1996). About 50 percent of non-Hodgkin's patients, having the low-grade indolent form, treated with this regimen showed responses. Therapeutic responses have also been obtained using 131I-labeled B1 (tositumomab) anti-CD20 murine monoclonal antibody when provided as repeated doses with pretreatment of unlabeled antibodies exceeding 600 mg per injection. Kaminski et al., N. Engl. J. Med. 329:459 (1993); Press et al., N. Engl. J. Med. 329:1219 (1993); Press et al., Lancet 346:336 (1995). However, these antibodies, whether provided as unconjugated forms or radiolabeled forms, have not shown high rates of objective and durable responses in patients with the more prevalent and lethal form of B-cell lymphoma, the intermediate or aggressive types. Therefore, a need exists to develop an immunotherapy for B-cell malignancies that achieves a therapeutic response of significant duration.
Additional studies targeting CD20 surface antigen have been performed using an anti-CD20 murine monoclonal antibody, IF5, which was administered by continuous intravenous infusion to B-cell lymphoma patients. Extremely high levels (>2 grams) of IF5 were reportedly required to deplete circulating tumor cells, and the results were described as being “transient.” Press et al., “Monoclonal Antibody IF5 (Anti-CD20) Serotherapy of Human B-Cell Lymphomas.” Blood 69/2:584-591 (1987). However, a potential problem with this approach is that non-human monoclonal antibodies (e.g., murine monoclonal antibodies) typically lack human effector functionality, i.e., they are unable to mediate complement-dependent lysis or lyse human target cells through antibody-dependent cellular toxicity or Fc-receptor mediated phagocytosis. Furthermore, non-human monoclonal antibodies can be recognized by the human host as a foreign protein and, therefore, repeated injections of such foreign antibodies can lead to the induction of immune responses leading to harmful hypersensitivity reactions. For murine-based monoclonal antibodies, this is often referred to as a Human Anti-Mouse Antibody (HAMA) response.
The use of chimeric antibodies is preferred because they do not elicit as strong a HAMA response as murine antibodies. Chimeric antibodies are antibodies which comprise portions from two or more different species. For example, Liu, A. Y. et al, “Production of a Mouse-Human Chimeric Monoclonal Antibody to CD20 with Potent Fc-Dependent Biologic Activity” J. Immunol. 139/10:3521-3526 (1987), describe a mouse/human chimeric antibody directed against the CD20 antigen. See also, PCT Publication No. WO 88/04936. An exemplary chimeric antibody would comprise mouse variable region sequences attached to human antibody constant region sequences.
The use of humanized antibodies is even more preferred, in order to further reduce the possibility of inducing a HAMA reaction. As discussed below, techniques for humanization of murine antibodies by replacing murine framework and constant region sequences with corresponding human antibody framework and constant region sequences are well known in the art and have been applied to numerous murine anti-cancer antibodies. Antibody humanization may also involve the substitution of one or more human framework amino acid residues with the corresponding residues from the parent murine framework region sequences.
Another approach that has improved the ability of antibodies to be effective in the treatment of B-cell disorders has been to conjugate a therapeutic agent, such as a radioactive or chemotherapeutic agent to the antibody, such that the agent is localized at the tumor site. For example, the above-referenced IF5 antibody and other B-cell antibodies have been labeled with 131I and were evaluated for biodistribution in two patients. See Eary, J. F. et al., “Imaging and Treatment of B-Cell Lymphoma” J. Nuc. Med. 31/8:1257-1268 (1990); see also, Press, O. W. et al., “Treatment of Refractory Non-Hodgkin's Lymphoma with Radiolabeled MB-1 (Anti-CD37) Antibody” J. Clin. Oncol. 7/8:1027-1038 (1989) (indication that one patient treated with 131I-labeled IF-5 achieved a partial response); Goldenberg, D. M. et al., “Targeting, Dosimetry and Radioimmunotherapy of B-Cell Lymphomas with 131I-Labeled LL2 Monoclonal Antibody” J. Clin. Oncol. 9/4:548-564 (1991) (three of eight patients receiving multiple injections reported to have developed a HAMA response to this CD22 murine antibody); Appelbaum, F. R. “Radiolabeled Monoclonal Antibodies in the Treatment of Non-Hodgkin's Lymphoma” Hem./Oncol. Clinics of N. Am. 5/5:1013-1025 (1991) (review article); Press, O. W. et al. “Radiolabeled-Antibody Therapy of B-Cell Lymphoma with Autologous Bone Marrow Support.” New England Journal of Medicine 329/17: 1219-12223 (1993) (131I-labeled anti-CD20 antibody IF5 and B1-tositumomab); and Kaminski, M. G. et al “Radioimmunotherapy of B-Cell Lymphoma with [131I] Anti-B1 (Anti-CD20) Antibody”. NEJM 329/7:459 (1993) (131I-labeled anti-CD20 antibody B1); PCT published application WO 92/07466 (antibodies conjugated to chemotherapeutic agents such as doxorubicin or mitomycin). However, these approaches have not eliminated the obstacles associated with using murine antibodies, despite the fact that many patients with lymphoma who have received prior aggressive cytotoxic chemotherapy are immune suppressed, thus having lower HAMA rates than lymphoma patients who have not been heavily pretreated.
Autoimmune diseases are a class of diseases associated with B-cell disorders. Examples comprise acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndromes, bullous pemphigoid, Type-I diabetes mellitus, Henoch-Schönlein purpura, post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitis obliterans, Sjögren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis and fibrosing alveolitis. The most common treatments are corticosteroids and cytotoxic drugs, which can be very toxic. These drugs also suppress the entire immune system, can result in serious infection, and have adverse affects on the bone marrow, liver and kidneys. There is a need for more effective methods of treating autoimmune diseases, particularly Class-III autoimmune diseases. A further need exists for the development of more effective antibodies for the treatment of cancer and/or autoimmune disease.
Still other diseases with immunological dysregulation are suitably treated by the novel compositions and methods described herein, such as hemolytic anemias, cryoglobulinemias, hepatitis (particularly hepatitis C), graft-versus-host disease (GVHD) (particularly after allogeneic stem cell transplantation), allosensitization (particularly with organ transplantation). There is now mounting evidence that B-cells are involved in these pathological states, so depleting B-cells by anti-B-cell therapies is gaining in interest (Roccatello et al., Clin Rev Allergy Immunol 2008, 34:111-117; Cutler et al., Blood 2006,108:756-752; Vo et al., N Engl J Med 2008, 359:242-251; Vieira et al., Transplantation 2004; 77:542-548; Abdallah & Prak, Clin. Transpl. 2006:427-37; Zaja et al., Bone Marrow Transplant., 2007, 40:273-77; Saadoun et al., Curr. Opin. Rheumatol. 2008, 20:23-8; Antonelli et al., Clin. Exp. Rheumatol. 2008, 26:S39-47).